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Research ArticleArticle

The Role of Human Nucleoside Transporters in Uptake of 3′-Deoxy-3′-fluorothymidine

Robert J. Paproski, Amy M. L. Ng, Sylvia Y. M. Yao, Kathryn Graham, James D. Young and Carol E. Cass
Molecular Pharmacology November 2008, 74 (5) 1372-1380; DOI: https://doi.org/10.1124/mol.108.048900
Robert J. Paproski
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Amy M. L. Ng
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Sylvia Y. M. Yao
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Kathryn Graham
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James D. Young
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Carol E. Cass
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Abstract

3′-Deoxy-3′-fluorothymidine (FLT) is a positron emission tomography (PET) tracer used to identify proliferating tumor cells. The purpose of this study was to characterize FLT transport by human nucleoside transporters (hNTs) and to determine the role of hNTs for FLT uptake in various human cancer cell lines. FLT binding to hNTs was monitored by the inhibitory effects of FLT on [3H]uridine uptake in yeast cells producing recombinant hNT proteins. hCNT1 displayed the lowest FLT Ki value for inhibition of [3H]uridine uptake, followed by hCNT3, hENT2, hENT1, and hCNT2. [3H]FLT was efficiently transported in Xenopus laevis oocytes individually producing hENT1, hENT2, hCNT1, or hCNT3. [3H]FLT uptake in MCF-7, A549, U251, A498, MIA PaCa-2, and Capan-2 cells was inhibited at least 50% by the hENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR). According to results of real-time polymerase chain reactions, hENT1 and hENT2 had the most abundant hNT transcripts in all cell lines. Cell lines also underwent 1) [3H]NBMPR equilibrium binding assays with or without 5-S-{2-(1-[(fluorescein-5-yl)thioureido]hexanamido)ethyl}-6-N-(4-nitrobenzyl)-5-thioadenosine, a membrane-impermeable NBMPR analog, to determine plasma membrane hENT1 levels, and 2) dose-response NBMPR inhibition of [3H]FLT uptake. MCF-7, A549, and Capan-2 cells displayed NBMPR IC50 values that were smaller or equal to NBMPR Kd values, suggesting that 50% inhibition of hENT1 reduced [3H]FLT uptake by at least 50%. A strong correlation between extracellular NBMPR binding sites/cell and [3H]FLT uptake was observed for all cell lines except MIA PaCa-2. These data suggest that plasma membrane hNTs (especially hENT1) are important determinants of cellular FLT uptake.

Footnotes

  • This work was supported by research grants from the National Cancer Institute of Canada, the Alberta Cancer Board, and the Canadian Institutes of Health Research. During the course of this work, R.J.P. was supported by the Endowed Graduate Studentship in Oncology: Recruitment Award and a studentship by the Alberta Heritage Foundation for Medical Research. C.E.C. was Canada Research Chair in Oncology and J.D.Y. is Heritage Scientist of the Alberta Heritage Foundation for Medical Research.

  • ABBREVIATIONS: PET, positron emission tomography; FLT, 3′-deoxy-3′-fluorothymidine; TK1, thymidine kinase 1; hNT, human nucleoside transporter; hENT, human equilibrative nucleoside transporter; hCNT, human concentrative nucleoside transporter; NBMPR, S-(p-nitrobenzyl)-6-thioinosine; NMDG, N-methyl-d-glucamine; CMM, complete minimal medium; GLU, glucose; FBS, fetal bovine serum; PCR, polymerase chain reaction; FTH-SAENTA, 5-S-{2-(1-[(fluorescein-5-yl)thioureido]hexanamido)ethyl}-6-N-(4-nitrobenzyl)-5-thioadenosine.

    • Received May 16, 2008.
    • Accepted July 30, 2008.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 74 (5)
Molecular Pharmacology
Vol. 74, Issue 5
1 Nov 2008
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Research ArticleArticle

The Role of Human Nucleoside Transporters in Uptake of 3′-Deoxy-3′-fluorothymidine

Robert J. Paproski, Amy M. L. Ng, Sylvia Y. M. Yao, Kathryn Graham, James D. Young and Carol E. Cass
Molecular Pharmacology November 1, 2008, 74 (5) 1372-1380; DOI: https://doi.org/10.1124/mol.108.048900

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Research ArticleArticle

The Role of Human Nucleoside Transporters in Uptake of 3′-Deoxy-3′-fluorothymidine

Robert J. Paproski, Amy M. L. Ng, Sylvia Y. M. Yao, Kathryn Graham, James D. Young and Carol E. Cass
Molecular Pharmacology November 1, 2008, 74 (5) 1372-1380; DOI: https://doi.org/10.1124/mol.108.048900
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